Novel reverse transcriptases and uses thereof

ABSTRACT

Hybrid reverse transcriptases formed from portions of FLVRT and MLVRT are provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present patent application is a divisional of U.S. patentapplication Ser. No. 15/895,504, filed Feb. 13, 2018, which claimsbenefit of priority to U.S. Provisional Patent Application No.62/459,974, filed on Feb. 16, 2017, each of which is incorporated byreference for all purposes.

REFERENCE TO A “SEQUENCE LISTING” SUBMITTED AS ASCII TEXT FILES VIAEFS-WEB

The Sequence Listing written in fileSequenceListing_094260-1243254-112720US.txt created on Mar. 22, 2021,184,785 bytes, machine format IBM-PC, MS-Windows operating system, inaccordance with 37 C.F.R. §§ 1.821- to 1.825, is hereby incorporated byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

The detection, analysis, transcription, and amplification of nucleicacids are frequently-used procedures in modern molecular biology. DNApolymerases are useful for detection and amplification of DNA or RNA.The application of such procedures for RNA analysis can involve theinvestigation of gene expression, diagnosis of infectious agents orgenetic diseases, and the generation of cDNA, to name but a fewapplications. The reverse transcription of RNA thus has many uses. Insome instances, the reverse transcriptase is followed by polymerasechain reaction amplification which can be used for rapid detection andquantification of RNA.

BRIEF SUMMARY OF THE INVENTION

In some embodiments, a hybrid reverse transcriptase is providedcomprising a finger domain, a palm domain, a thumb domain, a connectiondomain and an RNase H domain, wherein at least one of said domains is amouse leukemia virus reverse transcriptase (MLVRT) and other of saiddomains are from feline leukemia virus reverse transcriptase (FLVRT).

In some embodiments, the hybrid reverse transcriptase comprises: aportion of mouse leukemia virus reverse transcriptase (MLVRT) comprisingfinger and palm domains linked to a portion of feline leukemia virusreverse transcriptase (FLVRT) comprising thumb, connection, and RNase Hdomains. In some embodiments, the portion of the MLVRT has a carboxylterminus and the carboxyl terminus is linked directly to an aminoterminus of the portion of the FLVRT. In some embodiments, the portionof the MLVRT has a carboxyl terminus and the carboxyl terminus is linkedto an amino terminus of the portion of the FLVRT via a linking aminoacid sequence of 1-100 amino acids. In some embodiments, the portion ofthe MLVRT is at least 95% identical to SEQ ID NO:1 and the portion ofthe FLVRT is at least 95% identical to SEQ ID NO:5.

In some embodiments, the portion of the MLVRT comprises SEQ ID NO:1 andthe portion of the FLVRT comprises SEQ ID NO:5. In some embodiments, theportion of the FLVRT comprises SEQ ID NO:6 or SEQ ID NO:10. In someembodiments, the portion of the FLVRT comprises SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, or SEQ ID NO:11.

In some embodiments, the portion of the MLVRT comprises SEQ ID NO:2. Insome embodiments, the portion of the MLVRT comprises SEQ ID NO:3 or SEQID NO:4.

In some embodiments, the portion of the MLVRT comprises SEQ ID NO:4 andthe portion of the FLVRT comprises SEQ ID NO:7; or

the portion of the MLVRT comprises SEQ ID NO:4 and the portion of theFLVRT comprises SEQ ID NO:8; orthe portion of the MLVRT comprises SEQ ID NO:4 and the portion of theFLVRT comprises SEQ ID NO:9; orthe portion of the MLVRT comprises SEQ ID NO:3 and the portion of theFLVRT comprises SEQ ID NO:11; orthe portion of the MLVRT comprises SEQ ID NO:3 and the portion of theFLVRT comprises SEQ ID NO:10; orthe portion of the MLVRT comprises SEQ ID NO:4 and the portion of theFLVRT comprises SEQ ID NO:13.

In some embodiments, the hybrid reverse transcriptase comprises asequence substantially (e.g., at least 70%, 80%, 85%, 90%, or 95%)identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 34, or 35.

In some embodiments, the hybrid reverse transcriptase comprises aportion of feline leukemia virus reverse transcriptase (FLVRT)comprising finger and palm domains linked to a portion of mouse leukemiavirus reverse transcriptase (MLVRT) comprising RNase H domains. In someembodiments, the portion of MLVRT comprises thumb, connection, and RNaseH domains. In some embodiments, the portion of FLVRT comprises asequence at least 95% identical to SEQ ID NO:26; and the portion ofMLVRT comprises a sequence at least 95% identical to SEQ ID NO:28. Insome embodiments, the hybrid reverse transcriptase comprises a sequencesubstantially (e.g., at least 70%, 80%, 85%, 90%, or 95%) identical toSEQ ID NO:30.

In some embodiments, the portion of FLVRT comprises finger, palm, thumband connection domains. In some embodiments, the portion of FLVRTcomprises a sequence substantially (e.g., at least 70%, 80%, 85%, 90%,or 95%)identical to SEQ ID NO:27; and the portion of MLVRT comprises asequence substantially (e.g., at least 70%, 80%, 85%, 90%, or 95%)identical to SEQ ID NO:29. In some embodiments, the hybrid reversetranscriptase comprises a sequence substantially (e.g., at least 70%,80%, 85%, 90%, or 95%) identical to SEQ ID NO:31.

In some embodiments, the hybrid reverse transcriptase as described aboveor elsewhere herein has at least one mutation that improvesthermostability. In some embodiments, the at least one mutation at aposition corresponding to L139, D200, N479, D522, F526, H592, L601,E605, and H632 in SEQ ID NO:34.

Also provided is a nucleic acid comprising a polynucleotide encoding thehybrid reverse transcriptase as described above or elsewhere herein. Insome embodiments, the nucleic acid further comprises a (optionallyheterologous) promoter operably linked to the polynucleotide.

Also provided is an expression vector comprising the nucleic acid asdescribed above or elsewhere herein. Also provided is a cell comprisingthe expression vector. In some embodiments, the cell is a bacterialcell.

Also provided is a reaction mixture comprising: an RNA or DNA template;and the hybrid reverse transcriptase as described above or elsewhereherein. In some embodiments, the reaction mixture further comprises atleast one oligonucleotide primer and/or deoxynucleotides.

Also provided is a method of performing reverse transcription. In someembodiments, the method comprises contacting the hybrid reversetranscriptase as described above or elsewhere herein in a reactionmixture with a template RNA and a primer that hybridizes to the templateRNA under conditions such that the hybrid reverse transcriptase extendsthe primer in a template RNA-dependent manner to form a cDNA In someembodiments, the conditions comprise an extension step between 42-60° C.

Also provided is a kit comprising the hybrid reverse transcriptase asdescribed above or elsewhere herein. In some embodiments, the kitfurther comprises a DNA polymerase.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization described below are those well-known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. The techniques and procedures are generallyperformed according to conventional methods in the art and variousgeneral references (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well-known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

“Heterologous”, when used with reference to portions of a protein,indicates that the protein comprises two or more domains that are notfound in the same relationship to each other in nature. Such a protein,e.g., a fusion protein such as the hybrid RTs described herein, containstwo or more sequences covalently linked via a peptide bond or peptidelinker sequence arranged to make a new functional protein.

A “primer” refers to a polynucleotide sequence that hybridizes to asequence on a target nucleic acid and serves as a point of initiation ofnucleic acid synthesis. Primers can be of a variety of lengths and areoften less than 50 nucleotides in length, for example 12-30 nucleotides,in length. The length and sequences of primers for use in PCR can bedesigned based on principles known to those of skill in the art, see,e.g., Innis et al., supra.

“Polymerase” refers to an enzyme that performs template-directedsynthesis of polynucleotides. The term encompasses both the full lengthpolypeptide and a domain that has polymerase activity.

A “template” refers to a polynucleotide sequence that comprises thepolynucleotide to be amplified, optionally flanked by one or two primerhybridization sites.

As used herein, “nucleic acid” means DNA, RNA, single-stranded,double-stranded, or more highly aggregated hybridization motifs, and anychemical modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, electrostatic interaction,points of attachment and functionality to the nucleic acid ligand basesor to the nucleic acid ligand as a whole. Such modifications include,but are not limited to, peptide nucleic acids (PNAs), phosphodiestergroup modifications (e.g., phosphorothioates, methylphosphonates),2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases, isocytidine and isoguanidine and the like. Nucleicacids can also include non-natural bases, such as, for example,nitroindole. Modifications can also include 3′ and 5′ modifications suchas capping with a fluorophore (e.g., quantum dot) or another moiety.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e.,a carbon atom thatis bound to a hydrogen atom, a carboxyl group, an amino group, and an Rgroup, e.g., homoserine, norleucine, methionine sulfoxide, methioninemethyl sulfonium. Such analogs have modified R groups (e.g., norleucine)or modified peptide backbones, but retain the same basic chemicalstructure as a naturally occurring amino acid. Amino acid mimeticsrefers to chemical compounds that have a structure that is differentfrom the general chemical structure of an amino acid, but that functionsin a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “promoter” refers to regions or sequence located upstreamand/or downstream from the start of transcription and which are involvedin recognition and binding of RNA polymerase and other proteins toinitiate transcription.

A “vector” refers to a polynucleotide, which when independent of thehost chromosome, is capable replication in a host organism. Preferredvectors include plasmids and typically have an origin of replication.Vectors can comprise, e.g., transcription and translation terminators,transcription and translation initiation sequences, and promoters usefulfor regulation of the expression of the particular nucleic acid.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The terms “identical” or percent “identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence over acomparison window, as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Whenpercentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidsresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated according to, e.g., the algorithm of Meyers& Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

Sequences are “substantially identical” to each other if they have aspecified percentage of nucleotides or amino acid residues that are thesame (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region or whennot specified the whole sequence (SEQ ID NO)), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Accelrys), or by manual alignment and visualinspection.

Percent sequence identity and sequence similarity is determined usingthe BLAST algorithm, which is described in Altschul et al., J. Mol.Biol. 215:403-410 (1990). Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.go-v/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary soluble fraction SDS-PAGE gel following RTmutant expression.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that reverse transcriptase (RT) hybridsformed from a mouse leukemia virus reverse transcriptase (MLVRT) andfeline leukemia virus reverse transcriptase (FLVRT) have improvedsolubility compared to FLVRT enzymes. The hybrids described herein arealso expected to have improved stability, expression, or a combinationthereof compared to at least one of non-hybrid MLVRT or FLVRT enzymes.For example, the inventors have generated hybrid reverse transcriptasescomprising a finger domain, a palm domain, a thumb domain, a connectiondomain and an RNase H domain, wherein at least one of said domains is amouse leukemia virus reverse transcriptase (MLVRT) and other of saiddomains are from feline leukemia virus reverse transcriptase (FLVRT). Asdiscussed in more detail, the inventors have generated hybrids in whichthe finger and palm domains are either FLVRT or MLVRT sequences with atleast some of the remainder being from the alternative enzyme.

Polypeptides

Provided herein are hybrid reverse transcriptases (RTs) that have thefive RT domains (from amino to carboxyl: finger, palm, thumb,connection, and RNase H domains) where at least one (e.g., 1, 2, 3, or4) of those domains are from MLVRT and the remaining domain(s) are fromFLVRT. The structure of MLVRT and finger, palm, thumb, connection, andRNase H domains are described in, e.g., Das and Georgiadis, Structure12:819-829 (2004). The resulting hybrid RTs have improved expression(e.g., in E. coli) compared to an RT where all of the domains are fromFLVRT (i.e., wildtype FLVRT) while in some embodiments having improvedaccuracy compared to MLVRT.

MLV-FLV Hybrids

In some embodiments, the hybrid RT comprises the finger and palm domainsof MLVRT linked to the thumb, connection, and RNase H domains of FLVRT.Exemplary portions of MLVRT that comprise finger and palm domainsinclude, for example, SEQ ID NO:1 or a substantially identical sequencethereof. In some embodiments, the portion of MLVRT that comprises fingerand palm domains comprises SEQ ID NO:2 or a sequence substantiallyidentical thereto. The above-described MLVRT portion can be linked to aportion of FLVRT that comprises the thumb, connection, and RNase Hdomains. An exemplary portion of FLVRT that comprises the thumb,connection, and RNase H domains is SEQ ID NO: 5 or a substantiallyidentical sequence thereof. In some embodiments, the portion of FLVRTthat comprises the thumb, connection, and RNase H domains is SEQ ID NO:6 or a substantially identical sequence thereof or SEQ ID NO:10 or asubstantially identical sequence thereof. Exemplary hybrid RTs cancomprise, for example SEQ ID NO:1 or SEQ ID NO:2 any of SEQ ID NO:5, SEQID NO:6, or SEQ ID NO:10. In some embodiments, the hybrid RT comprisesone of SEQ ID NOs: 14, 15, 16, 17, 18, or 19 or a substantiallyidentical sequence thereof.

FLV-MLV Hybrids

In some embodiments, the hybrid RT comprises at least the finger andpalm domains of FLVRT linked to the thumb and connection domains ofeither FLVRT or MLVRT, which in turn is linked to the RNase H domains ofMLVRT. In some embodiments, the hybrid RT comprises finger and palmdomains of FLVRT and thumb, connection, and RNase H domains of MLVRT.Exemplary portions of FLVRT that comprise finger and palm domainsinclude, for example, SEQ ID NO:26 or a substantially identical sequencethereof. Exemplary portions of MLVRT that comprise thumb, connection,and RNase H domains include, for example, SEQ ID NO:28 or asubstantially identical sequence thereof.

In some embodiments, the hybrid RT comprises finger, palm, thumb, andconnection domains of FLVRT and RNase H domain of MLVRT. Exemplaryportions of FLVRT that comprise finger, palm, thumb, and connectiondomains include, for example, SEQ ID NO:27 or a substantially identicalsequence thereof. Exemplary portions of MLVRT that comprise the RNase Hdomain include, for example, SEQ ID NO:29 or a substantially identicalsequence thereof. In some embodiments, the hybrid RT comprises one ofSEQ ID NO: 30 or 31, or a substantially identical sequence thereof.

Any of the hybrid RTs described herein can include further amino acidsat the amino or carboxyl terminus. Exemplary additional amino acidsequences can include, for example, epitope tags or other tags thatallow for purification of the proteins or whose underlying codons allowfor cloning sites. Such tags can be fused at either end of the hybrid RTas most convenient for purification. Examples of such tags include, butare not limited to poly-histidine sequences or FLAG-tag. Various linkersequences can also be include to link such tags or other sequences tothe hybrid RT sequence. Linkers can include, for example glycine, serineor other amino acids that do not significantly interfere with proteinfolding such that the activity of the hybrid RT is not harmed. Thelinker sequences can also include protease cleavage sequences such thatthe tag can be removed by a protease or other cleavage mechanism,optionally once the hybrid RT has been purified (e.g., using the tag).In some embodiments, the hybrid RT includes one or more (e.g., 2-20,2-5, e.g., 3) alanines at the carboxyl terminus.

Thermostable Mutations

As noted herein, any hybrid RTs as described herein can include one ormore mutation that improves the thermostability (i.e., ability to remainactive during or after exposure to temperatures over 37° C., e.g.,42-60° C.) of the enzyme. Exemplary mutations include one or more (e.g.,2, 3, 4, 5, 6, or more) mutation at a position corresponding to L139(including but not limited to L139P), D200 (including but not limited toD200N), N479 (including but not limited to N479D), D522 (including butnot limited to, D522G, D522N, or D522A), F526 (including but not limitedto F526I), H592 (including but not limited to H592K), L601 (includingbut not limited to L601W), E605 (including but not limited to E605K),and H632 (including but not limited to H632Y) in SEQ ID NO:34. It shouldbe understood that such position designations do not indicate the numberof amino acids in the claimed molecule per se, but indicate where in theclaimed molecule the residue occurs when the claimed molecule sequenceis maximally aligned with SEQ ID NO:34.

Linking of Portions

Two portions of different RTs as described herein as described can bejoined via a linker by methods well known to those of skill in the art.These methods can include either recombinant and chemical methods.

Linking portions of different RTs may also comprise a peptide bondformed between moieties that are separately synthesized by standardpeptide synthesis chemistry or recombinant methods. Alternatively, insome embodiments, the coding sequences of each portion in the hybrid RTare directly joined and expressed as a fusion protein. Alternatively, anamino acid linker sequence may also be encoded in the polypeptide codingsequence and employed to separate the first and second RT portions by adistance sufficient to ensure that each polypeptide folds into itssecondary and tertiary structures. Such an amino acid linker sequence isincorporated into the fusion protein using recombinant techniques wellknown in the art. Suitable peptide linker sequences may be chosen basedon the following factors: (1) their ability to adopt a flexible extendedconformation; (2) their inability to adopt a secondary structure thatcould interact with functional epitopes on the first and secondpolypeptides; and (3) the lack of hydrophobic or charged residues thatmight react with the polypeptide functional epitopes. Typical peptidelinker sequences contain Gly, Ser, Val and Thr residues. Other nearneutral amino acids, such as Ala can also be used in the linkersequence. Amino acid sequences which may be usefully employed as linkersinclude those disclosed in Maratea et al. (1985) Gene 40:39-46; Murphyet al. (1986) Proc. Natl. Acad. Sci. USA 83:8258-8262; U.S. Pat. Nos.4,935,233 and 4,751,180. The linker sequence may generally be from 1 toabout 50 amino acids in length, e.g., 3, 4, 6, or 10 amino acids inlength, but can be 100 or 200 amino acids in length. Linker sequencesare not necessarily required.

Chemical linking can be performed, for example, as described inBioconjugate Techniques, Hermanson, Ed., Academic Press (1996). Joiningcan include, for example, derivitization for the purpose of linking thetwo proteins to each other, either directly or through a linkingcompound, by methods that are well known in the art of proteinchemistry. For example, in one chemical conjugation embodiment, themeans of linking the catalytic domain and the nucleic acid bindingdomain comprises a heterobifunctional-coupling reagent which ultimatelycontributes to formation of an intermolecular disulfide bond between thetwo moieties. Other types of coupling reagents that are useful in thiscapacity for the present invention are described, for example, in U.S.Pat. 4,545,985. Alternatively, an intermolecular disulfide mayconveniently be formed between cysteines in each moiety, which occurnaturally or are inserted by genetic engineering. The means of linkingmoieties may also use thioether linkages between heterobifunctionalcrosslinking reagents or specific low pH cleavable crosslinkers orspecific protease cleavable linkers or other cleavable or noncleavablechemical linkages. Other chemical linkers include carbohydrate linkers,lipid linkers, fatty acid linkers, polyether linkers, e.g., PEG, etc.For example, poly(ethylene glycol) linkers are available from ShearwaterPolymers, Inc. Huntsville, Alabama. These linkers optionally have amidelinkages, sulfhydryl linkages, or heterobifunctional linkages. Thelinking group can be a chemical crosslinking agent, including, forexample, succinimidyl-(N-maleimidomethyl)-cyclohexane-1-carboxylate(SMCC). The linking group can also be an additional amino acidsequence(s), including but not limited to, for example, a polyalanine,polyglycine or similarly, linking group.

In some embodiments, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thesequence. Non-classical amino acids include, but are not limited to, theD-isomers of the common amino acids, α-amino isobutyric acid,4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxy-proline, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, Cα-methyl amino acids, N-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

Expression and Purification

Nucleic acids encoding the hybrid RTs can be obtained using routinetechniques in the field of recombinant genetics. Basic texts disclosingthe general methods of use in this invention include Sambrook andRussell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001);Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); andCurrent Protocols in Molecular Biology (Ausubel et al., eds.,1994-1999). Such nucleic acids may also be obtained through in vitroamplification methods such as those described herein and in Berger,Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No.4,683,202; PCR Protocols A Guide to Methods and Applications (Innis etal., eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim& Levinson (Oct. 1, 1990) C & EN 36-47; The Journal Of NIH Research(1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173;Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell etal. (1989) J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu andWallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117,each of which is incorporated by reference in its entirety for allpurposes and in particular for all teachings related to amplificationmethods.

One of skill will recognize that modifications can additionally be madeto the hybrid RTs without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of a domain into a fusion protein. Such modifications arewell known to those of skill in the art and include, for example, theaddition of codons at either terminus of the polynucleotide that encodesthe binding domain to provide, for example, a methionine added at theamino terminus to provide an initiation site, or additional amino acids(e.g., poly His) placed on either terminus to create convenientlylocated restriction sites or termination codons or purificationsequences.

The hybrid RT polypeptides as described herein can be expressed in avariety of host cells, including E. coli, other bacterial hosts, yeasts,filamentous fungi, and various higher eukaryotic cells such as the COS,CHO and HeLa cells lines and myeloma cell lines. Techniques for geneexpression in microorganisms are described in, for example, Smith, GeneExpression in Recombinant Microorganisms (Bioprocess Technology, Vol.22), Marcel Dekker, 1994. Examples of bacteria that are useful forexpression include, but are not limited to, Escherichia, Enterobacter,Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus,Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.Filamentous fungi that are useful as expression hosts include, forexample, the following genera: Aspergillus, Trichoderma, Neurospora,Penicillium, Cephalosporium, Achlya, Podospora, Mucor, Cochliobolus, andPyricularia. See, e.g., U.S. Pat. No. 5,679,543 and Stahl and Tudzynski,Eds., Molecular Biology in Filamentous Fungi, John Wiley & Sons, 1992.Synthesis of heterologous proteins in yeast is well known and describedin the literature. Methods in Yeast Genetics, Sherman, F., et al., ColdSpring Harbor Laboratory, (1982) is a well-recognized work describingthe various methods available to produce the enzymes in yeast.

There are many expression systems for producing the polypeptides thatare well known to those of ordinary skill in the art. (See, e.g., GeneExpression Systems, Fernandex and Hoeffler, Eds. Academic Press, 1999;Sambrook and Russell, supra; and Ausubel et al, supra.) Typically, thepolynucleotide that encodes the polypeptide is placed under the controlof a promoter that is functional in the desired host cell. Manydifferent promoters are available and known to one of skill in the art,and can be used in the expression vectors of the invention, depending onthe particular application. Ordinarily, the promoter selected dependsupon the cell in which the promoter is to be active. Other expressioncontrol sequences such as ribosome binding sites, transcriptiontermination sites and the like are also optionally included. Constructsthat include one or more of these control sequences are termed“expression cassettes.” Accordingly, the nucleic acids that encode thejoined polypeptides are incorporated for high level expression in adesired host cell.

Expression control sequences that are suitable for use in a particularhost cell are often obtained by cloning a gene that is expressed in thatcell. Commonly used prokaryotic control sequences, which are definedherein to include promoters for transcription initiation, optionallywith an operator, along with ribosome binding site sequences, includesuch commonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056),the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.(1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad.Sci. U.S.A. (1983) 80:21-25); and the lambda-derived PL promoter andN-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128).The particular promoter system is not critical; any available promoterthat functions in prokaryotes and provides the desired level of activitycan be used. Standard bacterial expression vectors include plasmids suchas pBR322-based plasmids, e.g., pBLUESCRIPT™, pSKF, pET23D, lambda-phagederived vectors, and fusion expression systems such as GST and LacZ.Epitope tags can also be added to recombinant proteins to provideconvenient methods of isolation, e.g., c-myc, HA-tag, 6-His tag, maltosebinding protein, VSV-G tag, anti-DYKDDDDK (SEQ ID NO:42) tag, or anysuch tag, a large number of which are well known to those of skill inthe art.

The polypeptides described herein can be expressed intracellularly, orcan be secreted from the cell. Intracellular expression often results inhigh yields. If necessary, the amount of soluble, active fusionpolypeptide may be increased by performing refolding procedures (see,e.g., Sambrook et al., supra.; Marston et al., Bio/Technology (1984) 2:800; Schoner et al., Bio/Technology (1985) 3: 151). Polypeptides can beexpressed in a variety of host cells, including E. coli, other bacterialhosts, yeast, and various higher eukaryotic cells such as the COS, CHOand HeLa cells lines and myeloma cell lines. The host cells can bemammalian cells, insect cells, or microorganisms, such as, for example,yeast cells, bacterial cells, or fungal cells.

Once expressed, the polypeptides can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, gel electrophoresis and thelike (see, generally, R. Scopes, Protein Purification, Springer-Verlag,N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc. N.Y. (1990)). Substantially purecompositions of at least about 90 to 95% homogeneity are preferred, and98 to 99% or more homogeneity are most preferred. Once purified,partially or to homogeneity as desired, the polypeptides may then beused (e.g., as immunogens for antibody production).

To facilitate purification of the polypeptides, the nucleic acids thatencode the polypeptides can also include a coding sequence for anepitope or “tag” for which an affinity binding reagent is available.Examples of suitable epitopes include the myc and V-5 reporter genes;expression vectors useful for recombinant production of fusionpolypeptides having these epitopes are commercially available (e.g.,Invitrogen (Carlsbad Calif.) vectors pcDNA3.1/Myc-His andpcDNA3.1/V5-His are suitable for expression in mammalian cells).Additional expression vectors suitable for attaching a tag to the fusionproteins of the invention, and corresponding detection systems are knownto those of skill in the art, and several are commercially available(e.g., “FLAG” (Kodak, Rochester N.Y.). Another example of a suitable tagis a polyhistidine sequence, which is capable of binding to metalchelate affinity ligands. Typically, six adjacent histidines are used,although one can use more or less than six. Suitable metal chelateaffinity ligands that can serve as the binding moiety for apolyhistidine tag include nitrilo-tri-acetic acid (NTA) (Hochuli, E.(1990) “Purification of recombinant proteins with metal chelatingadsorbents” In Genetic Engineering: Principles and Methods, J. K.Setlow, Ed., Plenum Press, N.Y.; commercially available from Qiagen(Santa Clarita, Calif.)).

After biological expression or purification, the hybrid RTpolypeptide(s) may possess a conformation substantially different thanthe native conformations of the constituent polypeptides. In this case,it may be necessary or desirable to denature and reduce the polypeptideand then to cause the polypeptide to re-fold into the preferredconformation. Methods of reducing and denaturing proteins and inducingre-folding are well known to those of skill in the art (See, Debinski etal. (1993) J. Biol. Chem. 268: 14065-14070; Kreitman and Pastan (1993)Bioconjug. Chem. 4: 581-585; and Buchner et al. (1992) Anal. Biochem.205: 263-270). Debinski et al., for example, describe the denaturationand reduction of inclusion body proteins in guanidine-DTE. The proteinis then refolded in a redox buffer containing oxidized glutathione andL-arginine.

V. Methods of Use

Reverse transcription (RT) is an amplification method that copies RNAinto DNA. RT reactions can be performed with reaction mixtures asdescribed herein. For example, the invention provides for reversetranscribing one or more RNA (including for example, all RNA in a cell,e.g., to make a cDNA library) under conditions to allow for reversetranscription using a hybrid RT as described herein and generation of afirst and optionally second strand cDNA. The RT reaction can be primedwith a random primer, an oligo dT, or an RNA-specific primer. Componentsand conditions for RT reactions are generally known.

If desired, the reactions can further comprise RT-PCR. Standardtechniques for performing PCR assays are known in the art (PCRTechnology: Principles and Applications for DNA Amplification (Erlich,ed., 1989); PCR Protocols: A Guide to Methods and Applications (Innis,Gelfland, Sninsky, &, White, eds., 1990); Mattila et al., Nucleic AcidsRes. 19: 4967 (1991); Eckert & Kunkel, PCR Methods and Applications 1:17 (1991); Wallace et al., Ligase Chain Reaction, in Technologies forDetection of DNA Damage and Mutations, pp. 307-322 (Pfiefer, ed.,1996)). RT and PCR reactions are often used in the same assay and arereferred to as RT-PCR. RT-PCR combines reverse transcription of RNA intoDNA and subsequent DNA amplification reactions in a single reaction.Optimal reverse transcription, hybridization, and amplificationconditions will vary depending upon the sequence composition andlength(s) of the primers and target(s) employed, and the experimentalmethod selected by the practitioner. Various guidelines may be used toselect appropriate primer sequences and hybridization conditions (see,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.)(1989); Ausubel, F. M. et al., eds. (1999) Short Protocols in MolecularBiology, 4th edition, John Wiley & Sons); Ausubel, F. M. et al., eds.(1999-2010) Current Protocols in Molecular Biology, John Wiley & Sons).

In some embodiments, hybrid RTs described herein are used in a reversetranscriptase reaction at a higher temperature than would ordinarily beused. Thus, in embodiments, the some hybrid RTs described herein can beused at, 37° or 42° C., or a temperature greater than 42° C., forexample, between 42°-60°, 43°-55°, 45°-56°, 45°-65° C., etc. Highertemperature RT reactions are particularly helpful in situations wherethe template RNA forms secondary structure at normal RT temperatures(e.g., 37° or 42° C.) that partially or completely inhibit reversetranscription.

VI. Reaction Mixtures

Reaction mixtures comprising the hybrid RT polypeptides described hereinare provided. The reaction mixtures can comprise, for example, a targetnucleic acid, e.g., an RNA target where reverse transcription is to takeplace. The reaction mixtures can comprise appropriate nucleotides (e.g.,deoxynucleotides (dNTPs) or dideoxynucleotides) and in some embodiments,at least one buffer. Exemplary buffers can include, for example andwithout limitation, Tris, HEPES, ACES, PIPES, MOPSO, BES, MOPS, TES,TAPSO, POPSO, BICINE, TAPS, or AMPSO. The reaction mixtures canoptionally comprise one or more oligonucleotides that function as aprimer for template-dependent nucleic acid extension, one or moreoligonucleotides that function as a probe (e.g., linked to a label suchas a quencher, fluorescent dye, etc.), and/or a double stranded DNAbinding dye (e.g., SYBRGREEN). In some embodiments, the reaction mixturewill further comprises a separate DNA-dependent DNA polymerase. In someembodiments, the reaction mixture will further comprises magnesium(Mg++).

VII. Kits

In one aspect, kits for conducting reverse transcription (and optionallycyclic amplification, e.g., such as PCR) reactions are provided. In someembodiments, such kits include a hybrid RT as described herein, andoptionally dNTPs, and at least one buffer. Such kits may also includestabilizers and other additives to increase the efficiency of theamplification reactions. Such kits may also include one or more primers(e.g. poly-T, random hexamers, or specific primers) as well asinstructions for conducting reverse transcription reactions using thecomponents of the kits. In some embodiments, the kits will furthercomprises a separate DNA-dependent DNA polymerase.

EXAMPLES

In order to increase the recombinant FLV RT solubility in E. coli cells,hybrid RTs were constructed with part of the RT polypeptide sequencefrom MLV RT, and part of the RT sequence from FLV RT. Hybrid RTconstructs made exhibited improved solubility in E.coli cells asdemonstrated in FIG. 1.

The hybrid RT FM1 (⅓ FLV-⅔ MLV RT) included a N-terminal sequence of FLVRT from amino acid 1-279, which includes the finger and palm domains,and a C-terminal sequence of MLV RT from amino acid 280-671, whichincludes the thumb, connection and RNase H domain.

The hybrid RT FM2 (⅔ FLV-⅓ MLV RT) included a N-terminal sequence of FLVRT from amino acid 1-497, which includes the finger, palm, thumb, andconnection domains, and a C-terminal sequence of MLV RT from amino acid498-671, which includes the RNase H domain.

The hybrid RT MF includes a N-terminal sequence of MLV RT from aminoacid 1-277, which includes the finger and palm domains, and a C-terminalsequence of FLV RT from amino acid 276-667, which includes the thumb,connection, and RNase H domains.

The hybrid RT MF(P) includes a N-terminal sequence of MLV RT from aminoacid 1-221, which includes the finger and palm domains, and a C-terminalsequence of FLV RT from amino acid 221-667, which includes the thumb,connection, and RNase H domains.

Point mutations were introduced into MF and MF(P) hybrid RTs in order toimprove the enzyme performance.

Method of Expression of Recombinant RT Constructs:

Fresh LB broth was inoculated with overnight culture of BL21 cellscontaining expression plasmids in a ratio of 100:1. The cultures weregrown at 25° C. for about 6 hr or until OD600 nm=0.6-0.8. IPTG was addedto 0.1 mM, and grown O/N for 16 hrs at 16° C. Cells were harvested bycollecting the pellet after centrifugation. Cells were resuspended in200 ml lysis buffer and lysed by sonication. The cell debris was spundown and 200 μl of supernatant was collected. 5 μl sample and 5 μlloading buffer were combined in a PCR strip and heated at 95° C. for 5min. 6 ul of the samples were loaded onto an SDS-PAGE gel for analysis.Exemplary results are shown in FIG. 1.

The hybrid and mutant proteins were tested for a number ofcharacteristics, which is summarized in part in the following table(blanks indicate the activity was not tested):

Reaction Expression Thermostability Speed Processivity Level MF5PNAIYC++++ ++++ MF5PNAIY ++++ ++++ ++++ ++++ MF4GC ++++ ++++ MF4G ++++ ++++++++ ++++ MF(P)4GC ++++ ++++ MF(P)4G ++++ +++ ++++ ++++ FF4G +++ ++++++++ ++ FF4GC +++ ++ MF6PAIKYC ++++ ++++ FF4NDKW ++ +++ + ++ MF(P)4NDKW++ ++++ ++ ++++ MF3AIY +++ ++++ ++++ ++++ MF4CH ++++ ++++ ++++ ++++FF4CH +++ ++++ ++ ++ FF4C +++ ++++ ++ ++ MF(P)4C ++++ ++++ +++ ++++FF4NGWN ++ ++++ ++ ++ MF(P)4NGWN ++ ++++ ++ ++++ MD524G +++ ++++ ++++++++ MF(P)4C, FF4C: All “C” at the end represents an additional 15 aminoacid C-terminus native sequence from FLV RT. MF4CH , FF4CH: The “H” atthe end represents a histidine Tag at the C-terminus in addition to aN-terminus His-Tag. FF4G, FF4GC “FF” represents Feline RT mutants notthe fusion between MLV and FLV RT. FF4NDKW, FLV RT and Fusion RT thathave 4 point mutations at D200N, MF(P)4NDKW N477D, H592K, L601W.FF4NGWN, FLV RT and Fusion RT that have 4 point mutations at D200N,MF(P)4NGWN D522G, L601W, D651N.

A listing of exemplary mutant and hybrid sequences is provided below:

1. NF5PNAIYBased on FP(M)-TCH(F), which has finger and palm domains from MLVRT and thumb, connection and RNase H domain from FLV RT. Aminoacids at the following positions were mutated: L139P(160), D200N(221)D522A(543), F526I(547), H632Y(653). Italics indicate sequence from MLV;bolded text indicates sequence from FLV. (SEQ ID NO: 20)MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG P PPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQ RWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTAGSSIIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIYCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILAAA 2. MF4GCBased on FP(M)-TCH(F), which has finger and palm domains from MLVRT and thumb, connection and RNase H domain from FLV RT with an extendedC-terminal sequence. Amino acids at the following positions were mutated:L139P(160), D200N(221), D522G(543), L601W(622), E605K(626). Italicsindicate sequence from MLV; bolded text indicates sequence from FLV.(SEQ ID NO: 21)MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGL P PPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQ RWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSL N PATLLPLPSGKPPRLSPDLAETMAQTDLTDQP LPDADLTWYT GGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRG W LTS K GKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPKRP PWEYAAA3. MF4GBased on FP(M)-TCH(F), which has finger and palm domains from MLV RTand thumb, connection and RNase H domain from FLV RT without an extendedC-terminal sequence. Amino acids at the following positions were mutated:L139P(160), D200N(221), D522G(543), L601W(622), E605K(626). Italicsindicate sequence from MLV; bolded text indicates sequence from FLV.(SEQ ID NO: 22)MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGL P PPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQ RWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQP LPDADLTWYT GGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRG W LTS K GKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILAAA 4. MF(P)4GBased on FP(M)^(PstI)-TCH(F), which has a shorter finger and palmdomains sequence from MLV RT compared to MF4G. The thumb, connectionand RNase H domains from FLV RT without an extended C-terminalsequence. Amino acids at the following positions were mutated: L139P(160),D200N(221), D522G(543), L601W(622), E605K(626). Italics indicate sequencefrom MLV; bolded text indicates sequence from FLV. (SEQ ID NO: 23)MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG P PPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF N EALHRDLADFRIQHPDLILLQ YVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQT DLTDQPLPDADLTWYTG GSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRG W LTS K GKRIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILAAA 5. MF(P)4NDKWBased on FP(M)^(PstI)-TCH(F), which has a shorter finger and palmdomains sequence from MLV RT compared to MF4G. The thumb, connectionand RNase H domains from FLV RT without an extended C-terminalsequence. Amino acids at the following positions were mutated: D200N(221),N479D(500), H592K(613), L601W(622). Italics indicate sequence from MLV;bolded text indicates sequence from FLV. (SEQ ID NO: 24)MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF N EALHRDLADFRIQHPDLILLQ YVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSL D PATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHV K GEIYRRRG W LTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILAAA 6. MF(P)4NGWNBased on FP(M)^(PstI)-TCH(F), which has a shorter finger and palmdomains sequence from MLV RT compared to MF4G. The thumb, connectionand RNase H domains from FLV RT without an extended C-terminalsequence. Amino acids at the following positions were mutated: D200N(221),D522G(543), L601W(622), D651N(672). Italics indicate sequence from MLV;bolded text indicates sequence from FLV. (SEQ ID NO: 25)MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF N EALHRDLADFRIQHPDLILLQ YVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQT DLTDQPLPDADLTWYTG GSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRG W LTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLA N DTAKKAATETQSSLTILAAA 7. NF5PNAIYCBased on FP(M)-TCH(F), which has finger and palm domains from MLV RTand thumb, connection and RNase H domain from FLV RT with an extendedC-terminal sequence. Amino acids at the following positions were mutated:L139P(160), D200N(221), D522A(543), F526I(547), H632Y(653). Italicsindicate sequence from MLV; bolded text indicates sequence from FLV.(SEQ ID NO: 39)MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQ RWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTAGSSIIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSHIIYCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPKRPPW EYAAA8. MF4(P)GCBased on FP(M)^(PstI)-TCH(F), which has a shorter finger and palmdomains sequence from MLV RT compared to MF4G. The thumb, connectionand RNase H domains from FLV RT with an extended C-terminal sequence.Amino acids at the following positions were mutated: L139P(160),D200N(221), D522G(543), L601W(622), E605K(626). Italics indicatesequence from MLV; bolded text indicates sequence from FLV.(SEQ ID NO: 40)MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQ YVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIE GPKRPPWEYAAA9. FM1 (⅓ FLV-⅔ MLV RT)A hybrid RT with ⅓ of the N-terminal sequence from FLV RT(finger and palm domains) and the rest of ⅔ of the C-terminalsequence from MLV RT (Thumb, connection, RNase H domains). Italicsindicate sequence from MLV; bolded text indicates sequence from FLV.(SEQ ID NO: 30) MTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRWILDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQR WLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHDCLDILAEAHGTRSDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRAMADQAAREVATRETPGTSTLL 10. FM2 (⅔ FLV-⅓ MLV RT)A hybrid RT with ⅔ of the N-terminal sequence from FLV RT(finger, palm, thumb, connection domains) and the rest of ⅓ ofthe C-terminal sequence from MLV RT (RNase H domain only). Italicsindicate sequence from MLV; bolded text indicates sequence from FLV.(SEQ ID NO: 31) MTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRWILDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPD ILAEAHGTRSDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNMADQAAREVATRETPGTSTLL 11. Wild type MLV RT sequence(SEQ ID NO: 32)TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNMADQAARKAAITETPDTSTLL12. Wild type FLV RT sequence (SEQ ID NO: 33)TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTIL

Summary of Sequences:

-   SEQ ID NO:1: generic Short MLV-   SEQ ID NO:2: generic Longer MLV-   SEQ ID NO:3: specific Short MLV-   SEQ ID NO:4: specific Longer MLV-   SEQ ID NO:5: generic Shorter w/o c-term extension FLV-   SEQ ID NO:6: with longer c-term extension FLV: generic-   SEQ ID NO:7: Shorter w/o c-term extension FLV: specific mutations at    522, 526, 632-   SEQ ID NO:8: with longer c-term extension FLV: specific mutations at    522, 601, 605-   SEQ ID NO:9: Shorter w/o c-term extension FLV: specific mutations at    522, 601, 605-   SEQ ID NO:10: long N-term w/o c-term extension FLV: generic-   SEQ ID NO:11: long N-term w/o c-term extension FLV: specific    mutations at 522, 601, 605-   SEQ ID NO:12: long N-term w/o c-term extension FLV: specific    mutations at 479, 592, 601-   SEQ ID NO:13: long N-term w/o c-term extension FLV: specific    mutations at 522, 601, 651-   SEQ ID NO:14: NF5PNAIY without leader or end sequences-   SEQ ID NO:15: MF4GC without leader or end sequences-   SEQ ID NO:16: MF4G without leader or end sequences-   SEQ ID NO:17: MF4(P)G without leader or end sequences-   SEQ ID NO:18: MF(P)4NDKW without leader or end sequences-   SEQ ID NO:19: MF(P)4NGWN without leader or end sequences-   SEQ ID NO:20: NF5PNAIY with leader and end sequences-   SEQ ID NO:21: MF4GC with leader and end sequences-   SEQ ID NO:22: MF4G with leader and end sequences-   SEQ ID NO:23: MF(P)4G with leader and end sequences-   SEQ ID NO:24: MF(P)4NDKW with leader and end sequences-   SEQ ID NO:25: MF(P)4NGWN with leader and end sequences-   SEQ ID NO:26: ⅓ FLV N-terminus-   SEQ ID NO: 27 ⅔ FLV N-terminus-   SEQ ID NO:28 ⅔ MLV C-terminus-   SEQ ID NO:29 ⅓ MLV C-terminus-   SEQ ID NO:30 FM1 (⅓FLV-⅔ MLV RT)-   SEQ ID NO:31 FM2 (⅔FLV-⅓ MLV RT)-   SEQ ID NO: 32 Wild type MLV RT sequence-   SEQ ID NO: 33 Wild type FLV RT sequence-   SEQ ID NO:34: NF5PNAIYC without leader or end sequences-   SEQ ID NO:35: MF4(P)GC without leader or end sequences-   SEQ ID NO:36: NF5PNAIYC MLVRT portion-   SEQ ID NO:37: NF5PNAIYC FLVRT portion-   SEQ ID NO:38: MF4(P)GC MLVRT portion-   SEQ ID NO:39: MF4(P)GC FLVRT portion-   SEQ ID NO:40: NF5PNAIYC with leader and end sequences-   SEQ ID NO:41: MF4(P)GC with leader and end sequences

The examples and embodiments described herein are for illustrativepurposes only and that various modifications or changes in light thereofwill be suggested to persons skilled in the art and are to be includedwithin the spirit and purview of this application and scope of theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

SEQUENCES SEQ ID NO: 1: generic Short MLV:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG(L/P)PPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF(D/N)EALHRDLADFRIQHPDLILLQSEQ ID NO: 2: generic Longer MLV:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG(L/P)PPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF(D/N)EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQSEQ ID NO: 3: specific Short MLV:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQSEQ ID NO: 4: specific Longer MLV:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQSEQ ID NO: 5: generic Shorter w/o c-term  extension FLV:RWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYT(D/G/N/A)GSS(F/I)IRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHV(H/K)GEIYRRRG(L/W)LTS(E/K)GKEIKNKNEILALLEALFLPKRLSII(H/Y)CPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSEQ ID NO: 6: with longer c-term extension FLV: genericRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYT(D/G/N/A)GSS(F/I)IRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHV(H/K)GEIYRRRG(L/W)LTS(E/K)GKEIKNKNEILALLEALFLPKRLSII(H/Y)CPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPK RPPWEYSEQ ID NO: 7: Shorter w/o c-term extension FLV: specific 522, 526, 632RWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTAGSSIIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIYCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSEQ ID NO: 8: with longer c-term extension FLV: 522, 601, 605RWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPKRPPWEYSEQ ID NO: 9: Shorter w/o c-term extension FLV: specific 522, 601, 605RWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSEQ ID NO: 10: long N-term w/o c-term extension FLV: genericYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYT(D/G/N/A)GSS(F/I)IRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHV(H/K)GEIYRRRG(L/W)LTS(E/K)GKEIKNKNEILALLEALFLPKRLSII(H/Y)CPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSEQ ID NO: 11: long N-term w/o c-term extension FLV:specific 522, 601, 605YVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSEQ ID NO: 12: long N-term w/o c-term extension FLV:specific 479, 592, 601YVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLDPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVKGEIYRRRGWLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSEQ ID NO: 13: long N-term w/o c-term extension FLV:specific 522, 601, 651YVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLANDTAKKAATETQSSLTILSEQ ID NO: 14: NF5PNAIY without leader or end sequences:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTAGSSIIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIYCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTIL SEQ ID NO: 15: MF4GC without leader or end sequences:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPKRPPWEYSEQ ID NO: 16: MF4G without leader or end sequences:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTIL SEQ ID NO: 17: MF4(P)G without leader or end sequences:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGEAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSEQ ID NO: 18: MF(P)4NDKW without leader or end sequences:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGEAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLDPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVKGEIYRRRGWLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSEQ ID NO: 19: MF(P)4NGWN without leader or end sequences:YVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLANDTAKKAATETQSSLTILSEQ ID NO: 20: NF5PNAIY with leader and end sequences:MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTAGSSIIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIYCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILAAASEQ ID NO: 21: MF4GC with leader and end sequences:MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPKRPPWEYAAASEQ ID NO: 22: MF4G with leader and end sequences:MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILAAASEQ ID NO: 23: MF(P)4G with leader and end sequences:MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILAAASEQ ID NO: 24: MF(P)4NDKW with leader and end sequences:MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLDPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVKGEIYRRRGWLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILAAASEQ ID NO: 25: MF(P)4NGWN with leader and end sequences:MGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLANDTAKKAATETQSSLTILAAA SEQ ID NO: 26: ⅓ FLV N-terminusMTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWSEQ ID NO: 27 ⅔ FLV N-terminusMTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPD SEQ ID NO: 28 ⅔ MLV C-terminusLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHDCLDILAEAHGTRSDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREVATRETPGTSTLLSEQ ID NO: 29 ⅓ MLV C-terminusILAEAHGTRSDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREVATRETPGTSTLLSEQ ID NO: 30 FM1 (⅓ FLV-⅔ MLV RT)MTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHDCLDILAEAHGTRSDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREVATRETPGTSTLL SEQ ID NO: 31 FM2 (⅔ FLV-⅓ MLV RT)MTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGEAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDILAEAHGTRSDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREVATRETPGTSTLL SEQ ID NO: 32 Wild type MLV RT sequenceTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL SEQ ID NO: 33 Wild type FLV RT sequenceTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGEAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTIL SEQ ID NO: 34: NF5PNAIYC without leader or end sequences:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTAGSSIIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIYCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPKRPPWEYSEQ ID NO: 35: MF4(P)GC without leader or end sequences:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPKRPPWEY SEQ ID NO: 36: NF5PNAIYC MLVRT portion:MTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALEIRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQSEQ ID NO: 37: NF5PNAIYC FLVRT portion:RWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTAGSSIIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIYCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPKRPPWEYSEQ ID NO: 38: MF4(P)GC MLVRT portionMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQSEQ ID NO: 39: MF4(P)GC FLVRT portionYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPKRPPWEYSEQ ID NO: 40: NF5PNAIYC with leader and end sequencesMGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLEAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQ RWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTAGSSIIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIYCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIEGPKRPPW EYAAASEQ ID NO: 41: MF4(P)GC with leader and end sequencesMGSSHHHHHHSSGLVPRGSHMTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGINDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG1PPSHQWYTVLDLKDAFFCLRLHPTSQPLEAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFAEALHRDLADFRIQHPDLILLQ YVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTGGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGWLTSKGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILPTELIE GPKRPPWEYAAA

What is claimed is:
 1. A hybrid reverse transcriptase comprising afinger domain, a palm domain, a thumb domain, a connection domain and anRNase H domain, the hybrid reverse transcriptase comprising: a portionof feline leukemia virus reverse transcriptase (FLVRT) comprising thefinger domain and the palm domain, wherein the portion of the FLVRT isat least 95% identical to SEQ ID NO:26, said portion of the FLVRT linkedto a portion of mouse leukemia virus reverse transcriptase (MLVRT)comprising the thumb domain, the connection domain, and the RNase Hdomain, wherein the portion of the MLVRT is at least 95% identical toSEQ ID NO:28; or a portion of feline leukemia virus reversetranscriptase (FLVRT) comprising the finger domain, the palm domain, thethumb domain, and the connection domain wherein the portion of the FLVRTis at least 95% identical to SEQ ID NO:27, said portion of the FLVRTlinked to a portion of mouse leukemia virus reverse transcriptase(MLVRT) comprising the RNase H domain, wherein the portion of the MLVRTis at least 95% identical to SEQ ID NO:29.
 2. The hybrid reversetranscriptase of claim 1, a portion of feline leukemia virus reversetranscriptase (FLVRT) comprising the finger domain and the palm domain,wherein the portion of the FLVRT is at least 95% identical to SEQ IDNO:26, said portion of the FLVRT linked to a portion of mouse leukemiavirus reverse transcriptase (MLVRT) comprising the thumb domain, theconnection domain, and the RNase H domain, wherein the portion of theMLVRT is at least 95% identical to SEQ ID NO:28.
 3. The hybrid reversetranscriptase of claim 2, wherein the hybrid reverse transcriptasecomprises SEQ ID NO:30.
 4. The hybrid reverse transcriptase of claim 1,a portion of feline leukemia virus reverse transcriptase (FLVRT)comprising the finger domain, the palm domain, the thumb domain, and theconnection domain wherein the portion of the FLVRT is at least 95%identical to SEQ ID NO:27, said portion of the FLVRT linked to a portionof mouse leukemia virus reverse transcriptase (MLVRT) comprising theRNase H domain, wherein the portion of the MLVRT is at least 95%identical to SEQ ID NO:29.
 5. The hybrid reverse transcriptase of claim4, wherein the hybrid reverse transcriptase comprises SEQ ID NO:31. 6.The hybrid reverse transcriptase of claim 1, having at least onemutation that improves thermostability.
 7. A reaction mixturecomprising: an RNA or DNA template; and the hybrid reverse transcriptaseof claim
 1. 8. A method of performing reverse transcription, the methodcomprising contacting the hybrid reverse transcriptase of claim 1 in areaction mixture with a template RNA and a primer that hybridizes to thetemplate RNA under conditions such that the hybrid reverse transcriptaseextends the primer in a template RNA-dependent manner to form a cDNA.